The greatest potential for neutralization of HIV is before fusion of the virus with the target cell. HIV achieves membrane fusion via the action of just one protein, the trimeric gp120/gp41 envelope spike complex encoded by the Env gene. The viral spike is also the basis of antibody evasion by HIV, a process in which the hypervariable V1/V2 region of gp120 plays an important role. However, immunization with monomeric gp120 fails to elicit potent, broadly neutralizing antibodies. The search for an envelope-based HIV vaccine has, therefore, shifted to trimer-based vaccines. A detailed understanding of the epitope landscape presented by the viral spike is crucial for this goal but hindered by lack of structural information for the gp120/gp41 (gp140) trimer and V1/V2 region of monomeric gp120, which is highly exposed on trimer spike. I propose to determine the crystal structure of the gp140 trimer and a V1/V2-containing monomeric gp120 by X-ray crystallography. I will use fluorescence-detection size-exclusion chromatography (FSEC) of gp140- GFP and gp120-GFP fusion proteins to assess the stability, oligomerization state and molecular weight, and extent of monodispersity from nanogram quantities of unpurified material. This novel technique has expedited the crystallization of other difficult targets, including membrane proteins, and holds great promise for optimization of Env constructs. FSEC pre-crystallization screening combined with production of protein in transiently transfected insect Sf9 cells will allow me to rapidly screen multiple constructs of HIV-1 and SIV gp140 and gp120 and identify the most promising candidates for crystallization. High-throughput crystallization screening with automated Fluidigm Topaz microfluidic and RoboDesign CrystalMation instruments will be used to obtain crystals for structure determination. Structural characterization of the gp140 trimer and V1/V2-containing monomeric gp120 will greatly enhance our understanding of how HIV evades the immune system and provide critical information to the HIV vaccine design community. An HIV vaccine offers the best hope for controlling the AIDS epidemic, which, despite decades of research, shows no sign of abating.